Production of an immunoglobulin enriched fraction from whey protein solutions

ABSTRACT

This invention is directed to a process for producing an immunoglobulin enriched whey protein fraction from whey protein solutions. More particularly, it relates to the production of a whey protein fraction enriched in immunoglobulin, and optionally a whey protein isolate, using a cation exchanger under selected conditions. The selected conditions require overloading the cation exchanger with potentially absorbable protein which causes the exchanger to adsorb preferentially whey proteins other than immunoglobulin. The invention is also directed to the products produced by the process of the invention.

FIELD OF THE INVENTION

This invention relates to a process for producing an immunoglobulinenriched whey protein fraction from whey protein solutions. Moreparticularly, it relates to the production of a whey protein fractionenriched in immunoglobulins, and optionally a whey protein isolate,using a cation exchanger under selected conditions.

BACKGROUND OF THE INVENTION

Methods for isolating and purifying immunoglobulins from sourcematerials are well known in the art. For example, physical methods ofseparating immunoglobulins from serum are known. Separation may beeffected based on physical properties such as molecular weight,isoelectric point, electrophoretic mobility and solubility in varioussystems.

It is recognised that milk products contain, among other things, amixture of different proteins including immunoglobulin. A process forproducing immunoglobulin enriched milk products, and particularly thewhey left from cheese and casein making, is commercially desirable. Manymethods are known whereby whey proteins can be recovered from whey byion exchange either as mixtures of proteins or a particular proteinselected in preference to others.

Whether or not particular protein binds to an ion exchanger depends on alarge number of parameters including the isoelectric point (IEP) of thatprotein, the pH of the protein solution and especially the choice of ionexchanger. A protein which binds to a cation exchanger at a particularpH will not usually bind to an anion exchanger at the same pH. Theseeffects are well known to those skilled in the art and are summarised inTable 1 for the major whey proteins. It will be appreciated that this isan approximate guide to the behaviour of whey proteins on ion exchangersas there are also other factors which influence this behaviour.Furthermore, the information in the art has largely been determinedempirically.

TABLE 1 Conditions for Adsorption of Whey Proteins by Ion ExchangersCation Exchangers Anion Exchangers Protein IEP pH 3-5 pH 5-7 pH 3-5 pH5-7 Ig 6-8 Yes Yes No No α-LA, β-LG, BSA ≈5 Yes No No Yes GMP ≦4 No NoYes Yes

Immunoglobulin (Ig), α-Lactalbumin (α-LA), β-Lactoglobulin (β-LG),Bovine serum albumin (BSA), Glycomacropeptide (GMP present only in sweetwheys).

Given the isoelectric point of immunoglobulins of 6 to 8, their expectedbehaviour is that they will bind to cation exchangers at pH 3 to 7, butwill not bind to anion exchangers. Indeed, immunoglobulins are the mostdifficult of whey proteins to bind to an anion exchanger.

In keeping with this teaching, EP Patent No. 0320 152 discloses aprocess for producing a whey protein concentrate enriched inimmunoglobulins by contacting an anion exchanger with whey or wheyprotein concentrate at pH 5.5 to 7.0 preferably pH 6.0 to 6.4. Aftercontact with the exchanger, the unadsorbed protein fraction (effluent)contained a higher proportion of immunoglobulins than the original wheyor concentrate as a result of the preferential adsorption of the othermajor whey proteins.

It should also be noted that when sweet whey is used in the anionexchange process of EP 0 320 152 glycomacropeptide (GMP) can be adsorbedalong with the major whey proteins (as long as sufficient capacity isavailable), so the presence of GMP does not significantly affect thelevel of immunoglobulin enrichment.

It would be useful to have available an alternate process for producingan immunoglobulin enriched effluent using a cation exchanger.

Also consistent with this teaching are Japanese Patent No. 2104533 andBritish Patent No. 2179947 which describe processes wherein whey proteinsolutions are contacted with cation exchangers in the pH range 5-8 suchthat immunoglobulin is preferentially extracted by, and recovered inhigh purity from the ion exchanger. The utility of cation exchangers toadsorb immunoglobulins is further identified in GB 1,563,990 and FR2,452,881. These patents reflect the teachings in the art that cationexchangers are used to adsorb immunoglobulins.

In other published work with cation exchangers, where contact with wheyis made at lower pH e.g. 3-4.5, it has been reported that the proteinmixture called whey protein isolate (WPI), recovered from the cationexchanger contains the major whey proteins β-lactoglobulin,α-lactalbumin, BSA and immunoglobulins in the same ratio as in the wheyfrom which it was made. (Howell et al. Dairy Products TechnicalConference, 1990, pages 117-128, Wisconsin Centre for Dairy Research,Madison). Clearly immunoglobulins are usually adsorbed by the cationexchanger used to manufacture WPI.

All this is in keeping with the expected behaviour of whey proteins withion exchangers as set out in Table 1. In particular immunoglobulins donot bind easily to anion exchangers and so can be recovered at elevatedlevels from the effluent (breakthrough fraction). With cation exchangersthey are usually adsorbed and recovered from the ion exchanger with orwithout other proteins depending on the conditions used.

An exception to this is a report by A D A Kanekanian and M J Lewis (inDevelopments in Food Proteins, (B J F Hudson, Ed.) vol. 4, pages135-173, Elsivier, London 1986) that CM Cellulose adsorbed all theβ-lactoglobulin, α-lactalbumin and BSA from demineralised whey, at pH 3,but not the immunoglobulin fraction. However, the conditions identifiedeither are not specified in detail although they do indicate an underloading of the cation exchanger with adsorbable protein. Further, thereference teaches that ion-exchange processes are mainly useful forsolutions containing low concentrations of proteins (less than 1%) andthat column ion-exchange systems using both an anion and cationexchanger are preferred for use, the anion exchanger to bind most of thewhey proteins and the cation exchanger to bind the immunoglobulin.

Further, U.S. Pat. No. 4,834,994 discloses a method of removingβ-lactoglobulin selectively from whey by adsorption onto CM Celluloseunder very specific conditions of pH 4.3-4.6, 60-90% demineralisationand a protein concentration of 0.5 to 1.5%. The authors point out thatthe whey breakthrough stream has an increased ratio of α-lactalbumin toβ-lactoglobulin, because of the preferential adsorption and removal ofβ-lactoglobulin. The recovered β-lactoglobulin was reported ascontaining almost no immunoglobulin. The authors report that CMcellulose is not porous enough to bind immunoglobulin. However theapplicants have shown otherwise in Example 9.

Neither this patent nor the Lewis reference make any mention of thepossibility of producing a breakthrough fraction with enriched levels ofimununoglobulins. Furthermore, their methods may be limited to theirconditions of use.

NZ Patent No. 241328 also discloses a method of removing β-lactoglobulinfrom whey and more particularly producing an α-lactalbumin enrichedcation exchanger-passed solution (breakthrough) at a non-specifiedtemperature and under specific conditions which include a lengthycontact time of 20 hours (see Example 1).

No mention is made of whether the immunoglobulin finishes up with theβ-lactoglobulin or in the α-lactalbumin enriched stream. The latterwould make it enriched also in immunoglobulin. It cannot be assumed thatif α-lactalbumin does not neither will immunoglobulin. In fact, theopposite might be assumed on the basis of Table 1, that theimmunoglobulin would bind with the β-lactoglobulin. The applicants haveshown (comparative Example 2) that under the conditions disclosed toproduce an α-lactalbumim enriched stream from whey, the immunoglobulinis spread into both products and that the level of enrichment and yieldis not useful.

What the applicants have now surprisingly found is that for cationexchangers, which normally adsorb immunoglobulin from whey, it ispossible to reduce or prevent this adsorption taking place by contactingthem with an excess of adsorbable protein. Under such conditions theyhave found that the immunoglobulin has difficulty binding and can berecovered as a breakthrough solution whose protein content is enrichedin immunoglobulin.

This is particularly so when the whey protein solution used has a higherprotein concentration than that found in whey such as an ultrafiltrationretentate or reconstituted WPC. The use of such concentrated solutionshas the added advantage of making it easier to contact an excess ofadsorbable protein with the cation exchanger and to allow shortercontact times.

The net result of this is a process with a high yield of immunoglobulinin the WPC and a significant level of enrichment.

It is therefore an object of the present invention to provide a processfor producing an immunoglobulin enriched fraction from whey proteinsolutions which goes some way towards achieving these desiderata, or toat least provide the public with a useful choice.

SUMMARY OF THE INVENTION

In a first aspect, the present invention provides a process forproducing an inmunoglobulin enriched whey protein fraction andoptionally a whey protein isolate (WPI), which process comprises:

(a) subjecting a whey protein solution to ion exchange using a cationexchanger at pH 2.5-4.5 under conditions which overload the cationexchanger with potentially adsorbable protein and which cause theexchanger to adsorb preferentially whey proteins other thanimmunoglobulin,

(b) recovering the breakthrough whey protein fraction not adsorbed bysaid ion exchange step (a); and optionally

(c) eluting and recovering WPI adsorbed to said cation exchanger.

In a further embodiment, the present invention may be broadly said toconsist in an immunoglobulin enriched whey protein fraction wheneverprepared by a process of the invention.

For the purposes of this specification the expression “to overload”means to load an ion exchanger with a quantity of potentially adsorbableprotein in excess of that which the ion exchanger is able to adsorb.This results in less than the maximum yield of adsorbable protein.

BRIEF DESCRIPTION OF THE DRAWINGS

The present invention will now be described in more detail withreference to the accompanying drawings in which;

FIG. 1 is a plot of protein capacity of ion exchanger against yield ofWPI showing the varying immunoglobulin G contents (% of protein) of adeproteinated UF retentate of a cheese whey stream at VCF 2.5 and themaximum yield possible for WPI.

FIG. 2 is a plot as in FIG. 1 but of deproteinated WPC obtainedinitially from H₂SO₄ casein whey.

FIG. 3 is a plot of WPI yield and immunoglobulin G content of WPC as apercentage of WPC protein against pH.

FIG. 4 is a plot similar to that of FIG. 1 and FIG. 2 showing the effectof VCF and dilution of retentate on the performance of ion exchanger.

DESCRIPTION OF THE INVENTION

The present invention relates to a process for producing animmunoglobulin enriched fraction from a whey protein solution.

The applicants have surprisingly found that for cation exchangers whichnormally adsorb immunoglobulin from whey, it is possible to reduce orprevent this absorption taking place by contacting the cation exchangerwith an excess of adsorbable protein that is, overloading the ionexchanger. Generally, a pH level below about 4.5 has been found to beadvantageous. It is these findings by the applicants which form thebasis for the present invention.

In one aspect, as defined above, the present invention provides aprocess for producing an immunoglobulin enriched protein fraction andoptionally a whey protein isolate (WPI) from a whey protein solution.The process involves the step (a) subjecting a whey protein solution toion exchange using a cation exchanger under conditions which overloadthe cation exchanger with potentially adsorbable protein and which causethe exchanger to adsorb preferentially whey proteins other thanimmunoglobulin.

The solution is contacted with the ion exchanger at a pH, temperature,and for a time which are sufficient to enable the whey proteins otherthan immunoglobulin to be adsorbed onto the ion exchanger. This step isfollowed by the step of recovering the breakthrough whey proteinfraction not adsorbed by the ion exchange step, and optionally therecovery of WPI adsorbed by the ion exchanger.

Whey protein solutions suitable for use in step (a) of the process ofthe invention include ultrafiltration retentate prepared from eithersweet wheys or acid wheys, and reconstituted whey protein concentrates.Concentrates may be prepared by ultrafiltration process known in theart. The whey protein solution may also be desalted by diafiltrationand/or ion exchange using an ion exchanger under conditions which doesnot adsorb immunoglobulin.

If desired defatting may also be effected using known separating,centrifuging or clarifying techniques.

A further optional pre-step is also possible and preferred for sweetwhey protein solutions. Removal of GMP from sweet whey further increasesthe immunoglobulin level. GMP may be removed in a pre-step byultrafiltration as pH ≦4 as taught in the art for example, in EP 393,850to produce GMP reduced retentate which is then used in the process ofthe present invention.

Alternatively, GMP may be removed in a pre-step by adjusting the pH ofthe whey or retentate from 6 to 4.6-5.0 and contacting it with an anionexchanger to selectively bind GMP as taught in the art, for example inGB 2,188,526. The pH is then further adjusted to from 2.5 to 4.5 andused in the process of the present invention. pH adjustments may beeffected by the addition of suitable acids such as hydrochloric acid,sulphuric acid, lactic acid, citric acid and acetic acid.

More preferably acid whey protein solution is used in the process of thepresent invention. Acid wheys do not have GMP in them to be left behindin the breakthrough stream and thereby dilute the immunoglobulin.Consequently, the immunoglobulin content of the breakthrough is muchhigher at 20-30% of residual protein compared with that obtained fromsweet wheys.

It is desirable that the whey protein solution is concentrated toproduce a retentate. The process of the invention proved more effectivewith a retentate than whey as illustrated in Table 2. A retentate can beachieved by subjecting the selected whey solution to ultrafiltration toachieve a volume concentration factor of from 2.5 to 25. Thisultrafiltration may optionally be followed by diafiltration. Suitableconditions and examples of ultrafiltration and diafiltration will beknown to those skilled in the art. For example, an ultrafiltrationmembrane with a nominal molecular weight cut off in the range of from5,000 Da to about 50,000 Da, more preferably 10,000 Da may be used.

Alternatively, a whey protein concentrate (WPC powder) may bereconstituted with an appropriate quantity of water to achieve thedesired volume concentration factor.

Further adjustments may be made including dilution with water, pHadjustments using acid, and reduction in ionic strength by H⁺ ionexchange with an ion exchanger (such as DOWEX™ 50W-X8 (H⁺) resin) whichdoes not adsorb protein. Generally, the lower the ionic strength thegreater the protein concentration that can be used. The reader'sattention is directed to our related specification PCT/NZ97/00005(incorporated herein by reference) for a more comprehensive discussionon protein concentrations.

As specified above, the ion exchange step in the process of the presentinvention is effected using a cation exchanger. The cation exchangerused in the process of the invention may be selected from suitableinorganic and organic cation exchangers known in the art. Preferably,the cation exchanger selected is an organic cellulose base cationexchanger. More preferably, the cellulose cation exchanger is a cationexchanger prepared from regenerated cellulose as disclosed in U.S. Pat.No. 4,175,183.

Suitable cellulose cation exchangers include CM, SP and SE exchangers.Generally, a strong acid type cation exchanger such as a sulphonic acid(for example, SP or SE) exchanger is preferred.

An SP cellulose cation exchanger, such as SP GIBCOCEL™ HG2, isparticularly preferred.

The ion exchange process of the present invention is carried out underconditions which cause the cation exchanger to preferentially adsorbwhey proteins other than immunoglobulin. It is not intended by thisexpression that the sum total of all whey proteins other thanimmunglobulin will be adsorbed.

The main proteins found in bovine whey and ion exchange conditionstraditionally considered useful for their recovery are detailed in Table1 above. It will be appreciated from this table that the conditionsimpacting on the adsorption of protein by the ion exchanger include pH,and as the applicants have found, the amount of adsorbable proteinapplied to the ion exchanger.

The overload conditions in process step (a) are preferably achieved byincreasing the ratio of the weight of whey protein in solution to thevolume of cation exchanger used so as to achieve a yield of WPI which isless than the maximum yield of adsorbable protein.

Preferably said yield is about 40 to 60% of total protein for sweetwheys and about 50 to 80% of total protein for acid wheys.

Alternatively, said yield is up to about 40% of total protein less thanthe maximum yield possible.

Viewed in another way, the ion exchanger is overloaded by supplying aquantity of adsorbable protein in excess of that which gives the maximumyield of adsorbed protein. The maximum yield can be readily determinedby profiling adsorption according to the techniques demonstrated in thefollowing examples.

The pH at which the process step (a) is conducted is also critical. ThepH range of 2.5 to 4.5 is selected. A preferred pH range is 3.0 to 4.2.The applicants have found that over this pH range the whey proteinsα-Lactoglobulin (α-LA), β-lactoglobulin (β-LG) and bovine serum albumin(BSA) are adsorbed in preference to immunoglobulin when the cationexchanger is overloaded with adsorbable protein.

The temperature at which the process is conducted may range from about5° C. to about 50° C., preferably less than 20° C. and most preferablyfrom 8° C. to 15° C. The lower temperatures are preferred to minimisethe growth of mesophilic bacteria and to prevent protein denaturationespecially of the immunoglobulin.

The step of contacting the whey solution with the ion exchanger can becarried out in any convenient manner. While contact in a columnexchanger is feasible, a stirred bed of ion exchanger is preferred foruse in the present invention.

Contact time with the ion exchanger may range from about 30 minutes upto 90 minutes at 10° C. Prior art processes using dilute proteinsolutions as found in whey such as that in NZ 241328, teach adsorptiontimes of up to 20 hours. The present applicants teach significantlyreduced contact times, particularly where concentrated whey proteinsolutions with reduced ionic strength are used.

In step (b) of the process of the invention the breakthrough wheyprotein fraction, not adsorbed by the ion exchange step (a), isrecovered. Recovery may be effected according to any suitable processknown in the art. For example, neutralisation, followed byultrafiltration and/or diafiltration. Further processing steps ofevaporation and freeze-drying or spray-drying may also be effected if adry product is required.

In a preferred process step (b), the breakthrough is first neutralisedto between pH 6 and 7. Neutralisation can be effected by addition of asuitable base such as sodium or potassium hydroxide. Concentration ofthe product is then effected by ultrafiltration. The resulting retentatemay then be further neutralised if required. Desirably, the retentate isthen spray dried to give an immunoglobulin enriched WPC powder.

Step (c) of the process of the invention provides for the elution andrecovery of the WPI from the cation exchanger. Although not essential,it is usually desirable from an economic point of view to recover boththe adsorbed protein and the breakthrough protein. The adsorbed protein(WPI) may be recovered according to elution, ultrafiltration, anddrying, techniques as known in the art and outlined herein.

In the presently preferred process, the ion exchanger with adsorbedprotein is washed with water and the protein eluted by a pH shift to 9by addition of a base such as sodium hydroxide. This pH shift isaccomplished preferably in a stirred slurry of water and exchanger.Elution is desirably carried for between 30 minutes and 3 hours, mostpreferably 1 hour at 10° C. However, a temperature range between 5° C.and 50° C. may be employed if desired. The eluate is then drained andthe resin again washed with water. This eluate solution is thendesirably ultrafiltrated and spray dried to give a WPI powder depletedin immunoglobulin.

In a further embodiment, the present invention also provides a wheyprotein fraction enriched in immunoglobulin whenever prepared by aprocess of the invention. Also forming a part of the invention is thewhey protein solution depleted in immunoglobulin as produced by aprocess of the invention. As discussed above, the products may be in theform of a breakthrough solution and eluted protein solutionrespectively, or may be further processed to provide dry powderproducts.

The products as produced by the present invention have applications innutritional and pharmaceutical products. The immunoglobulin enrichedfraction may be further formulated into feedstuffs and particularlyanimal feedstuffs. The immunoglobulin content may provide temporarypassive immunity as well as initiating the active immune system innewborns. This can increase disease resistance and increase growthrates.

The uses for the WPI product are well documented in the art. Theyinclude preparation of foodstuffs, nutritional products, and drinks anddietary supplements.

EXAMPLES

The following examples are intended to be illustrative only and in noway limit the scope of the present invention.

Example 1

Comparative Example

Cheese whey derived from a cheddar cheesemaking process was adjusted topH 3.5 with 2 M sulphuric acid. A 320 mL sample of this was mixed with50 mL of cation exchanger (SG GIBCOCEL™ HG2 cellulose manufactured byLife Technologies Ltd, New Zealand, Gibco BRL)¹ for one hour at 25° C.and then filtered on a sintered glass filter. The ion exchanger waswashed with water and the combined breakthrough of filtrate and washingswas made up to 450 mL. The breakthrough solution thus obtained wasanalysed for true protein by Kjeldahl nitrogen analyses and forimmunoglobulin G by FPLC using a Protein G column. Sixty-eight percentof the total protein and all of the imnmunoglobulin G was found to havebeen removed by the cation exchanger. These proteins were recovered fromthe ion exchanger by elution at pH 9 to give a whey protein isolatecontaining 8% immunoglobulin G.

¹ SP GIBCOCEL™ cellulose cation exchanger has a settled volume of 1.4mL/g of washed and drained product. It was previously known as “IndionS”.

Example 2

Comparative Example

Cheese whey was adjusted to pH 3.5 with 2 M sulphuric acid. Samples (350mL) were mixed with 17, 21, 28 and 42 mL lots of cation exchanger (SPGiboCel™ HG2) for one hour at 20° C. which maintaining the pH at 3.5 bythe further addition of 2 M sulphuric acid. The cation exchanger wasthen collected on a sintered glass filter and washed with water. Thefiltrate and washings were collected and made up to 400 mL. Thisbreakthrough solution was analysed as in Example 1. The starting wheywas similarly analysed. The results are shown in Table 2. Under theseconditions with whey, the immunoglobulin G in the deproteinised wheystream (breakthrough) does not increase usefully above that present inthe original whey even when the cation exchanger was contacted with alarge excess of protein (146 mg/mL) such that the adsorbed protein onlyamounted to 43% of the total present.

TABLE 2 Adsorption of Protein from Whey by SP GIBCOCEL ™ CelluloseCation Exchanger Volume of SP GIBCOCEL ™ Cellulose Cation ExchangerUsed, mL 17 21 28 42 Whey* used, g 350 350 350 350 Protein load, mg/mLof SP 146 117 87 58 IgG Yield in Breakthrough, % 65 65 58 41 IgG/Proteinin Breakthrough, % 6.1 7.1 7.2 5.8 *Whey: 0.70% protein and 5.4%IgG/Protein

Cheese whey was concentrated two and a half times by ultrafiltration.This retentate with a volume concentration factor of 2.5 (VCF 2.5) wasadjusted to pH 3.5 with 2 M sulphuric acid and 114 g samples of it weremixed with SP GIBCOCEL™ HG2 cellulose cation exchanger using 50, 38, 25,19 and 15 mL lots. After mixing for one hour at 25° C. the SP GIBCOCEL™cellulose cation exchanger was collected on a sintered glass filter,drained and washed with water. The combined filtrate and washings weremade up to a volume of 175 mL and analysed as described in Example 1.

FIG. 1 shows the maximum yield of WPI as being 68-70% (from thisparticular UF retentate from cheese whey) no matter how much ionexchanger is used to bind the protein. Under these conditions theimmunoglobulin G is also adsorbed and appears in the WPI product.

FIG. 1 also shows how the immunoglobulin G content of the protein in thebreakthrough solution from the ion exchanger increases as the yield ofWPI is decreased. The operating capacity of the cation exchangerincreases under these conditions. When the yield was limited to 50%there was an increase in the immunoglobulin G content of the retenitateto 11%, based on total protein, as compared with 6% in the feed to theion exchanger. Further concentration of this exchanger breakthroughsolution by ultrafiltration gave an 80% protein WPC which analysed ashaving an immunloglobulin G content of 10% of total solids.

Recovery of the protein from the SP GIBCOCEL™ cellulose cation exchangerby elution at pH 9 gave a WPI with less than 2% immunoglobulin G.

A WPC powder (80% protein) obtained from sulphuric acid casein whey wasreconstituted to give a 5.5% protein solution corresponding to a VCF 10retentate from ultrafiltration of whey. The pH of this was adjusted to3.8 with 2 M sulphuric acid and 20 mL amounts were mixed with 20, 13.3and 10 mL lots of SP GIBCOCEL™ cellulose cation exchanger HG2 for 30minutes at about 10° C. while maintaining the pH at 3.8. The exchangerwas then collected on a sintered glass filter, drained and washed withwater. The combined filtrate and washings were made up to a volume of 50mL and analysed as described in Example 1.

The results are presented in Table 3 and FIG. 2 and show that themaximum WPI yield is around 80% of total protein, when the SP GIBCOCEL™cellulose cation exchanger is loaded with less than 55 g/L. By doublingthe load, the yield of WPI decreases to around 70% but:

(i) the operating capacity of the SP resin improves dramatically andhence the cost of WPI production is reduced.

(ii) the recovery of immunoglobulin G in the breakthrough improvesdramatically and likewise its cost of production.

(iii) the immunoglobulin G content of the WPC stream is increased to 25%of protein present. This is significantly greater than the 9% found inthe original WPC from sulplhturic acid casein whey.

By overloading the ion exchanger with protein to decrease the yield ofWPI from about 80% to about 70%, and co-producing two products, weobtained these advantages.

TABLE 3 Conditions for Co-producing WPI and IgG-Enriched WPC from AcidWhey Retentate Load Protein Load SP Capacity WPI Yield Immunoglobulin G⁴Ratio¹ g/L² g/L² %³ Yield % Purity % 1:1 55 43 78 55 20 1:1:5 83 62 7569 25 1:2 110  76 69 84 25 ¹Ratio (v/v) of SP GIBCOCEL ™ cellulosecation exchanger; VCF 10 retentate ²g of protein per liter of SPGIBCOCEL ™ cellulose cation exchanger ³% of the total proteins in theretentate ⁴IgG remaining in the breakthrough stream.

Example 5

This was the same as Example 5 except that the pH was varied from 3 to4.2 using 20 mL of reconstituted WPC at 5.5% protein and mixing it with13.3 mL of SP GIBCOCEL™ cellulose cation exchanger. The yield of WPI andimmunoglobulin G content of the residual protein in the breakthroughstreams are shown in FIG. 3 which shows that it was possible to producean immunoglobulin G enriched WPC stream across the pH range 3.0 to 4.2.

Example 6

The same batch of cheese whey as used in Example 2 was concentrated byultrafiltration to give retentates with volume concentration factors(VCF) of about 12 and 23. These were used in place of whey as in Example2. The amount of retentate applied to the SP GIBCOCEL™ cellulose cationexchanger was correspondingly reduced to 28 and 15 g respectively sothat the total amount of protein presented to each lot of ion exchangerremained constant. In addition the VCF23 retentate was diluted withtwice its weight of water and 45 g used. Table 4 summarizes theconditions used and shows the yield and content of imunoglobulin G inthe non-adsorbed protein in the WPC stream. FIG. 4 shows the effect ofWPI yield and operating capacity of the ion exchange on thisimmunoglobulin G level.

TABLE 4 Absorption of Protein from Various Cheese Whey Retentates VCF12VCF23 VCF23 & H₂O Protein Conc, 9.11 15.1 35.2 % IgG/Protein, % 6.3 676.7 Weight used, g 28 15 45 SP 17 21 28 42 17 21 21 28 42 GibcoCel ™, mLProtein load, 150 121 91 61 133 108 111 84 56 mg/mL SP IgG yield 93 7263 36 85 74 71 53 41 in WPC, % IgG/Protein, % 11.4 10.1 9.9 6.7 11.010.8 12.8 10.5 8.6

Example 7

Twenty kg of WPC powder containing 80% protein (sold under the tradename ALACEN 392) manufactured from cheese whey was reconstituted at 20%total solids and its pH was adjusted to pH 3.75 with 10% sulphuric acid.This solution was mixed for 40 minutes at 10° C. with 130 L of SPGIBCOCEL™ HG2 cellulose cation exchange resin that was previouslyflooded with water. This mixing was carried out in a 500 L tank fittedwith a screen across the bottom. At the end of the mixing period theresin was drained and washed with 100 L of water.

The breakthrough and washings (335 L at 2.2% protein) were adjusted topH 6.2 with sodium hydroxide and concentrated to 16% total solids byultrafiltration on Koch HFK 131 membranes. The resulting letentate wasfurther neutralised and spray dried to give an Ig-enriched WPC powder.The immunoreactive Immunoglobulin G content of this powder (asdetermined by the radial immune diffusion method, Binding Site assaykit, Binding Site Ltd, Birminghain, UK), was 14.3% Kjeldahl protein ascompared with an IgG level of 8.8% of protein in the original cheeseWPC.

The adsorbed protein was recovered from the washed and drained resin byadding one bed volume of water and then mixing for one hour at 10° C. atpH 8.0 by the addition of sodium hydroxide. The elute was then drainedfrom the vessel and the resin was washed with approximately a bed volumeof water to give 290 L of elute at 2.7% protein. The elute solution wasultrafiltered and spray dried to give a whey protein isolate powder withan IgG content of only 3.2% of protein.

Example 8

Seventeen kg of a WPC powder containing approximately 80% protein (soldunder the trade name ALACEN 134) manufactured from acid casein whey wasreconstituted at 20% total solids and its pH was adjusted to pH 3.75with 10% sulphuric acid. This solution was mixed with a cation exchangerunder the same conditions as for Example 7.

The breakthrough and washings (330 l at 1.3% protein) were adjusted topH 6.2 with sodium hydroxide and concentrated to 15% total solids byultrafiltration on Koch HFK 131 membranes. The resulting retentate wasfurther neutralised and freeze dried to give an Ig-ennrched WPC powder.The immunoreactive Immunoglobulin G content of this powder (asdetermined by RID) was 23.9% of Kjeldahl protein as compared with an IgGlevel of 10.4% of protein in the original WPC.

WPC powder as used in Example 7 was reconstituted at 9% total solids andits pH adjusted to pH 3.8 with 10% sulphuric acid. This solution (30 g)was mixed for 30 minutes at 10° C. with 25.6 g (31 mL) of the CMcellulose, WHATMAN CM52™ in the sodium ion form, which had been washedwith water and drained under vacuum on a sintered glass filter. The pHwas maintained at 3.8 during this time and then the CM cellulose wascollected on the filter, drained and washed with water. The combinedfiltrate and washings (breakthrough) was then analysed for protein andimmunoglobulin G as described in Example 1. The reconstituted WPCsolution was similarly analysed. It was found that 52% of theimmunoglobulin G had been adsorbed by the CM cellulose.

Industrial Application

It is believed that the process of the present invention for producingan immunoglobulin enriched fraction from a protein solution will findwidespread acceptance in the dairy industry. The process is effectiveusing concentrated whey protein solutions, enabling lower batch volumesand shorter contact times with the cation exchanger. More generally, thecollection of two useful fractions at the same time has benefits of bothreducing plant effluent produced and providing better economics ofscale. The process is also a useful alternative to methods of collectingimmunoglobulin enriched fractions as a WPI.

The immunoglobulin enriched products produced by the process of theinvention have utility in nutritional and pharmaceutical products,particularly animal feedstuffs. Feedstuffs with increased immunoglobulincontent may provide temporary passive immunity to an animal as well asinitiating the active immune system in newborns. This increases diseaseresistance and increases growth rates.

It will be appreciated by those persons skilled in the art although thepresent invention has been described with reference to specificembodiments, modifications and alterations of the embodiments describedcan be made without departing from the scope of the invention.

What is claimed is:
 1. A process for producing an immunoglobulinenriched whey protein fraction and optionally a whey protein isolate(WPI), which process comprises: (a) subjecting a 1× to 23× concentratedwhey protein solution at a pH of 3.0 to 4.2 and at a temperature of 10°C. to 25° C. to conditions under which a sulfopropyl (SP) cationexchanger is overloaded with potentially adsorbable protein and whichcause the exchanger to adsorb whey proteins other than immunoglobulins;(b) recovering the whey protein fraction not adsorbed by said ionexchange step (a); and (c) optionally eluting and recovering the WPIabsorbed to said cation exchanger.
 2. A process as claimed in claim 1wherein the WPI is eluted and recovered.
 3. A process as claimed inclaim 1 wherein the whey protein solution is an ultrafiltrationretentate.
 4. A process as claimed in claim 3 wherein theultrafiltration retentate has a volume concentration factor of 10 or 23.5. A process as claimed in claim 3 wherein the whey protein solution orretentate is reduced in ionic strength.
 6. A process as claimed in claim5 wherein the reduced ionic strength is achieved by diafiltration,dilution or H⁺ ion exchange with an ion exchanger.
 7. A process asclaimed in claim 2 wherein the weight of whey protein in solution to thevolume of cation exchanger used achieves a yield of whey protein isolate(WPI) which is less than the maximum yield of adsorbable protein.
 8. Aprocess as claimed in claim 7 wherein said yield is about 40% to 60% oftotal protein for sweet whey.
 9. A process as claimed in claim 7 whereinsaid yield is about 50% to 80% of total protein for acid whey.
 10. Aprocess as claimed in claim 7 wherein said yield is up to about 40% oftotal protein less than the maximum yield possible.
 11. A process asclaimed in claim 1 wherein the protein solution is contacted with thecation exchanger for less than about 2 hours.
 12. A process as claimedin claim 1 wherein said cation exchanger is a cellulose based cationexchanger.
 13. A process as claimed in claim 12 wherein said cationexchanger is regenerated cellulose cation exchanger.
 14. A process asclaimed in claim 1 wherein the recovered whey protein faction issubjected to ultrafiltration and the retentate recovered.
 15. A processas claimed in claim 14 wherein the retentate is spray-dried to produce adry whey protein concentrate.
 16. A process as claimed in claim 2wherein the WPI is subjected to ultrafiltration and the retentaterecovered.
 17. A process as claimed in claim 16 wherein the retentate isspray-dried to produce a dry WPI concentrate.